Protein-protein interaction of a pharaonis halorhodopsin mutant forming a complex with pharaonis halobacterial transducer protein ii detected by fourier-transform infrared spectroscopy

Pharaonis halorhodopsin (pHR) functions as a light-driven inward chloride ion pump in Natoronomonas pharaonis, while pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, pSRII), is a light sensor for negative phototaxis. ppR forms a 2:2 complex with its cognate transducer protein (pHtrII) through intramembranous hydrogen bonds: Tyr199^ppR-Asn74 ^pHtrII and Thr189^ppR-Glu43^pHtrII, Ser62 ^pHtrII. It was reported that a pHR mutant (P240T / F250Y), which possesses the hydrogen-bonding sites, impairs its pumping activity upon complexation with pHtrII. In this study, effect of the complexation with pHtrII on the structural changes upon formation of the K, L₁ and L ₂ intermediates of pHR was investigated by use of Fourier-transform infrared spectroscopy. The vibrational changes of Tyr250^pHR and Asn74^pHtrII were detected for the L₁ and L₂ intermediates, supporting that Tyr250^pHR forms a hydrogen bond with Asn74^pHtrII as similarly to Tyr199^ppR. The conformational changes of the retinal chromophore were never affected by complexation with pHtrII, but amide-I vibrations were clearly different in the absence and presence of pHtrII. The molecular environment around Asp156^pHR in helix D is also slightly affected. These additional structural changes are probably related to blocking of translocation of a chloride ion from the extracellular to the cytoplasmic side during the photocycle.